The present invention relates to an improved immunoassay method for determining carcinoembryonic antigen (CEA) in a serum sample, which corrects for false positives due to the presence in the sample of human anti-species antibodies.
It is well known to carry out immunoassays for antigens which may be present in the serum or other body fluids of a patient, in order to obtain diagnostically useful information. These immunoassays may use one or more polyclonal or monoclonal antibodies. The presence of heterophile antibodies in human serum has long been recognized. Heterophile antibodies are antibodies that bind to several species of non-human immunoglobulins. Some immunoassays include one or more animal sera as components of the diluent to coagulate heterophile antibodies, since these antibodies can interfere with assay.
It is also known to heat acidified human serum or plasma samples to coagulate interfering matrix protein, or "inert proteins". Hirai, Cancer Research, 37:2267-2274, 1977, disclosed pretreatment of serum samples for a dual polyclonal antibody radioimmunoassay (RIA) for CEA. Dilute serum samples, buffered at pH 5, were heated at 85.degree. C. for 10 minutes prior to immunoassay to avoid interference from inert proteins in serum. Kim et at., Clin. Chem., 25:773-776, 1979, disclosed heat treatment of plasma and serum samples at pH 5, at a temperature of 70.degree.-80.degree. C., for 10-20 minutes, prior to dual polyclonal antibody RIA, to coagulate "non-specific proteins" in the sample that interfered with CEA determination.
In an abstract distributed to attendees at the VIIth Meeting of the International Society for Oncodevelopmental Biology and Medicine, entitled Tumor Markers, held in Surrey, England, in September, 1979, Keep et al., abstract No. 176, disclosed that heat treatment of CEA-containing extracts of human tumors and of normal colon and meconium led to a reduction in measured CEA values, using a dual polyclonal antibody RIA and a dual antibody polyclonal enzyme immunoassay (EIA). The effect was reportedly masked by human serum components, however, and these authors reported that heat treatment of patient' sera at 85.degree. C. for 10 minutes did not diminish their CEA levels as measured by both RIA and EIA. The presence of "heat-labile and heat-stable components of CEA" was noted, and polyclonal antisera raised against heat-treated CEA were mentioned, as was a polyclonal antiserum that reacted predominantly with heat-labile CEA. These authors also noted that dilution of CEA with serum or immunoglobulin appeared to confer some protection to CEA with respect to heat treatment.
The recent trend to use of monoclonal antibodies, especially murine monoclonals from ascites production or raised in cell culture, in sandwich assays has led to a recognition that human anti-species antibodies in serum can sometimes interfere with such assays and lead to false positive results. For example, a sandwich assay for CEA in human serum or plasma, using a pair of murine monoclonal capture and probe antibodies, would show a false elevation in the presence of substantial amounts of human anti-mouse antibodies (HAMA), since such antibodies could bridge the capture and probe antibodies by binding of one arm of the anti-mouse antibody to each.
A need exists for a CEA immunoassay method which can include as an optional step a simple procedure for eliminating falsely elevated readings due to the presence of human anti-non-human species antibodies such as HAMA. Such an assay would be of particular value to clinicians who need to determine and monitor CEA levels in cancer patients, especially those who may have been treated or are being treated with murine monoclonal antibodies for tumor imaging or therapy. As other types of non-human monoclonal or polyclonal antibodies are developed for in vivo use, the method of the invention will serve a wider market.